Temet M. McMichael

Phosphorylation of the antiviral protein interferon-inducible transmembrane protein 3 (IFITM3) dually regulates its endocytosis and ubiquitination.

Background: IFITM3 restricts the fusion of viruses within endolysosomes. Results: Phosphorylation of IFITM3 on Tyr20 blocks IFITM3 endocytosis and ubiquitination. Conclusion: Tyr20 of IFITM3 is part of a YXXΦ endocytosis signal and has a dual role in regulating IFITM3 ubiquitination. Significance: Identifying mechanisms regulating IFITM3 trafficking and activity is crucial for understanding and manipulating IFITM3 biology for combating virus infections. Interferon-inducible transmembrane protein 3 (IFITM3) is essential for innate defense against influenza virus in mice and humans. IFITM3 localizes to endolysosomes where it prevents virus fusion, although mechanisms controlling its trafficking to this cellular compartment are not fully understood. We determined that both mouse and human IFITM3 are phosphorylated by the protein-tyrosine kinase FYN on tyrosine 20 (Tyr20) and that mouse IFITM3 is also phosphorylated on the non-conserved Tyr27. Phosphorylation led to a cellular redistribution of IFITM3, including plasma membrane accumulation. Mutation of Tyr20 caused a similar redistribution of IFITM3 and resulted in decreased antiviral activity against influenza virus, whereas Tyr27 mutation of mouse IFITM3 showed minimal effects on localization or activity. Using FYN knockout cells, we also found that IFITM3 phosphorylation is not a requirement for its antiviral activity. Together, these results indicate that Tyr20 is part of an endocytosis signal that can be blocked by phosphorylation or by mutation of this residue. Further mutagenesis narrowed this endocytosis-controlling region to four residues conforming to a YXXΦ (where X is any amino acid and Φ is Val, Leu, or Ile) endocytic motif that, when transferred to CD4, resulted in its internalization from the cell surface. Additionally, we found that phosphorylation of IFITM3 by FYN and mutagenesis of Tyr20 both resulted in decreased IFITM3 ubiquitination. Overall, these results suggest that modification of Tyr20 may serve in a cellular checkpoint controlling IFITM3 trafficking and degradation and demonstrate the complexity of posttranslational regulation of IFITM3.

Palmitoylation on Conserved and Nonconserved Cysteines of Murine IFITM1 Regulates Its Stability and Anti-Influenza A Virus Activity

ABSTRACT The interferon-induced transmembrane proteins (IFITMs) restrict infection by numerous viruses, yet the importance and regulation of individual isoforms remains unclear. Here, we report that murine IFITM1 (mIFITM1) is palmitoylated on one nonconserved cysteine and three conserved cysteines that are required for anti-influenza A virus activity. Additionally, palmitoylation of mIFITM1 regulates protein stability by preventing proteasomal degradation, and modification of the nonconserved cysteine at the mIFITM1 C terminus supports an intramembrane topology with mechanistic implications.

E3 Ubiquitin Ligase NEDD4 Promotes Influenza Virus Infection by Decreasing Levels of the Antiviral Protein IFITM3.

Interferon (IFN)-induced transmembrane protein 3 (IFITM3) is a cell-intrinsic factor that limits influenza virus infections. We previously showed that IFITM3 degradation is increased by its ubiquitination, though the ubiquitin ligase responsible for this modification remained elusive. Here, we demonstrate that the E3 ubiquitin ligase NEDD4 ubiquitinates IFITM3 in cells and in vitro. This IFITM3 ubiquitination is dependent upon the presence of a PPxY motif within IFITM3 and the WW domain-containing region of NEDD4. In NEDD4 knockout mouse embryonic fibroblasts, we observed defective IFITM3 ubiquitination and accumulation of high levels of basal IFITM3 as compared to wild type cells. Heightened IFITM3 levels significantly protected NEDD4 knockout cells from infection by influenza A and B viruses. Similarly, knockdown of NEDD4 in human lung cells resulted in an increase in steady state IFITM3 and a decrease in influenza virus infection, demonstrating a conservation of this NEDD4-dependent IFITM3 regulatory mechanism in mouse and human cells. Consistent with the known association of NEDD4 with lysosomes, we demonstrate for the first time that steady state turnover of IFITM3 occurs through the lysosomal degradation pathway. Overall, this work identifies the enzyme NEDD4 as a new therapeutic target for the prevention of influenza virus infections, and introduces a new paradigm for up-regulating cellular levels of IFITM3 independently of IFN or infection.

Regulation of the trafficking and antiviral activity of IFITM3 by post-translational modifications.

IFITM3 restricts cellular infection by multiple important viral pathogens, and is particularly critical for the innate immune response against influenza virus. Expression of IFITM3 expands acidic endolysosomal compartments and prevents fusion of endocytosed viruses, leading to their degradation. This small, 133 amino acid, antiviral protein is controlled by at least four distinct post-translational modifications. Positive regulation of IFITM3 antiviral activity is provided by S-palmitoylation, while negative regulatory mechanisms include lysine ubiquitination, lysine methylation and tyrosine phosphorylation. Herein, we describe specific insights into IFITM3 trafficking and activity that were provided by studies of IFITM3 post-translational modifications, and discuss evidence suggesting that IFITM3 adopts multiple membrane topologies involving at least one intramembrane domain in its antivirally active conformation.

IFITM3 requires an amphipathic helix for antiviral activity

Interferon‐induced transmembrane protein 3 (IFITM3) is a cellular factor that blocks virus fusion with cell membranes. IFITM3 has been suggested to alter membrane curvature and fluidity, though its exact mechanism of action is unclear. Using a bioinformatic approach, we predict IFITM3 secondary structures and identify a highly conserved, short amphipathic helix within a hydrophobic region of IFITM3 previously thought to be a transmembrane domain. Consistent with the known ability of amphipathic helices to alter membrane properties, we show that this helix and its amphipathicity are required for the IFITM3‐dependent inhibition of influenza virus, Zika virus, vesicular stomatitis virus, Ebola virus, and human immunodeficiency virus infections. The homologous amphipathic helix within IFITM1 is also required for the inhibition of infection, indicating that IFITM proteins possess a conserved mechanism of antiviral action. We further demonstrate that the amphipathic helix of IFITM3 is required to block influenza virus hemagglutinin‐mediated membrane fusion. Overall, our results provide evidence that IFITM proteins utilize an amphipathic helix for inhibiting virus fusion.

IFITMs from Mycobacteria Confer Resistance to Influenza Virus When Expressed in Human Cells.

Interferon induced transmembrane proteins (IFITMs) found in vertebrates restrict infections by specific viruses. IFITM3 is known to be essential for restriction of influenza virus infections in both mice and humans. Vertebrate IFITMs are hypothesized to have derived from a horizontal gene transfer from bacteria to a primitive unicellular eukaryote. Since bacterial IFITMs share minimal amino acid identity with human IFITM3, we hypothesized that examination of bacterial IFITMs in human cells would provide insight into the essential characteristics necessary for antiviral activity of IFITMs. We examined IFITMs from Mycobacterium avium and Mycobacterium abscessus for potential antiviral activity. Both of these IFITMs conferred a moderate level of resistance to influenza virus in human cells, identifying them as functional homologues of IFITM3. Analysis of sequence elements shared by bacterial IFITMs and IFITM3 identified two hydrophobic domains, putative S-palmitoylation sites, and conserved phenylalanine residues associated with IFITM3 interactions, which are all necessary for IFITM3 antiviral activity. We observed that, like IFITM3, bacterial IFITMs were S-palmitoylated, albeit to a lesser degree. We also demonstrated the ability of a bacterial IFITM to co-immunoprecipitate with IFITM3 suggesting formation of a complex, and also visualized strong co-localization of bacterial IFITMs with IFITM3. However, the mycobacterial IFITMs lack the endocytic-targeting motif conserved in vertebrate IFITM3. As such, these bacterial proteins, when expressed alone, had diminished colocalization with cathepsin B-positive endolysosomal compartments that are the primary site of IFITM3-dependent influenza virus restriction. Though the precise evolutionary origin of vertebrate IFITMs is not known, our results support a model whereby transfer of a bacterial IFITM gene to eukaryotic cells may have provided a selective advantage against viral infection that was refined through the course of vertebrate evolution to include more robust signals for S-palmitoylation and localization to sites of endocytic virus trafficking.

Human Genetic Determinants of Viral Diseases

Much progress has been made in the identification of specific human gene variants that contribute to enhanced susceptibility or resistance to viral diseases. Herein we review multiple discoveries made with genome-wide or candidate gene approaches that have revealed significant insights into virus-host interactions. Genetic factors that have been identified include genes encoding virus receptors, receptor-modifying enzymes, and a wide variety of innate and adaptive immunity-related proteins. We discuss a range of pathogenic viruses, including influenza virus, respiratory syncytial virus, human immunodeficiency virus, human T cell leukemia virus, human papilloma virus, hepatitis B and C viruses, herpes simplex virus, norovirus, rotavirus, parvovirus, and Epstein-Barr virus. Understanding the genetic underpinnings that affect infectious disease outcomes should allow tailored treatment and prevention approaches in the future.

The palmitoyltransferase ZDHHC20 enhances interferon-induced transmembrane protein 3 (IFITM3) palmitoylation and antiviral activity

Interferon-induced transmembrane protein 3 (IFITM3) is a cellular endosome- and lysosome-localized protein that restricts numerous virus infections. IFITM3 is activated by palmitoylation, a lipid posttranslational modification. Palmitoylation of proteins is primarily mediated by zinc finger DHHC domain–containing palmitoyltransferases (ZDHHCs), but which members of this enzyme family can modify IFITM3 is not known. Here, we screened a library of human cell lines individually lacking ZDHHCs 1–24 and found that IFITM3 palmitoylation and its inhibition of influenza virus infection remained strong in the absence of any single ZDHHC, suggesting functional redundancy of these enzymes in the IFITM3-mediated antiviral response. In an overexpression screen with 23 mammalian ZDHHCs, we unexpectedly observed that more than half of the ZDHHCs were capable of increasing IFITM3 palmitoylation with ZDHHCs 3, 7, 15, and 20 having the greatest effect. Among these four enzymes, ZDHHC20 uniquely increased IFITM3 antiviral activity when both proteins were overexpressed. ZDHHC20 colocalized extensively with IFITM3 at lysosomes unlike ZDHHCs 3, 7, and 15, which showed a defined perinuclear localization pattern, suggesting that the location at which IFITM3 is palmitoylated may influence its activity. Unlike knock-out of individual ZDHHCs, siRNA-mediated knockdown of both ZDHHC3 and ZDHHC7 in ZDHHC20 knock-out cells decreased endogenous IFITM3 palmitoylation. Overall, our results demonstrate that multiple ZDHHCs can palmitoylate IFITM3 to ensure a robust antiviral response and that ZDHHC20 may serve as a particularly useful tool for understanding and enhancing IFITM3 activity.

A balancing act between IFITM3 and IRF3

Interferon (IFN)-induced transmembrane protein 3 (IFITM3) is a potent antiviral factor capable of restricting numerous virus infections.1 Viruses inhibited by IFITM3 generally enter cells via endocytosis, and are trafficked to IFITM3positive intracellular membrane compartments.1,2 IFITM3-positive compartments are acidic and stain positive for a variety of endosomal, lysosomal and autophagosomal markers.2,3 Although the membrane fusion of many endocytic viruses, such as influenza virus, is triggered by acidic pH, fusion is blocked in the presence of IFITM3, and virions are degraded2,4,5 (Figure 1). It has been proposed that IFITM3 alters membrane properties, including rigidity and curvature, to inhibit virus membrane fusion.5 The molecular mechanism underlying these IFITM3-induced membrane alterations is not well characterized, and whether IFITM3 has other cellular functions is also not known. IFITM3 is particularly well characterized as a restriction factor able to limit influenza virus infections, and is responsible for a significant portion of the antiviral action of type I IFNs against influenza virus in cells.6,7 As such, IFITM3 knockout mice succumb to sublethal doses of influenza virus and exhibit increased lung pathology compared to control mice.8,9 Further, homozygosity of a single nucleotide polymorphism in the human IFITM3 gene, rs-12252-C, has been associated with increased morbidity and mortality in individuals hospitalized from infections with the 2009 pandemic H1N1 influenza A virus or with an emergent H7N9 virus.8,10,11 This polymorphism is thought to result in an alternatively spliced IFITM3 transcript coding for a truncated and mislocalized protein, though evidence for a truncated variant of IFITM3 is lacking.8 Nonetheless, IFITM3 is well established as a critical component of the innate anti-influenza virus immune response in mice and humans.1 Although IFITM3 is appreciated primarily as an antiviral protein able to block virus membrane fusion, we, and others, have also noted the ability of IFITM3 to induce autophagosome formation in cells as indicated by LC3 lipidation and puncta formation.2,3 We previously demonstrated that antiviral activity of IFITM3 is independent of the canonical ATG5-dependent autophagy pathway, thus decoupling autophagy induction by IFITM3 from its antiviral activity.3 This suggested that, in addition to directly inhibiting viruses, IFITM3 may also play a role in regulating autophagy, though the biological relevance of such a role has been difficult to identify. New work by Jiang et al.12 published in this edition of Cellular and Molecular Immunology, identified a novel function for IFITM3 in inhibiting IFN-β induction by promoting degradation of IFN regulatory factor 3 (IRF3), a critical transcription factor necessary for activating IFN-β production upon infection. The authors propose that this IRF3 degradation occurs within IFITM3associated autophagosomes (Figure 1). This work describes the first reported immunoregulatory role for IFITM3 in the type I IFN pathway. Jiang et al.12 made the astute observation that IFITM3 expression reduces production of IFN-β during Sendai virus infection (an IFITM3insensitive virus) or after PolyI:C treatment of cells. By artificially inducing IFN-β through overexpression of various components of the IFN-β induction pathway, they then determined that IFITM3 acts late in the pathway, possibly by affecting IRF3. They went on to show that indeed IRF3 levels inversely correlate with IFITM3 levels; when IFITM3 was overexpressed, IRF3 levels decreased, and when IFITM3 expression was knocked down, IRF3 levels increased. Interestingly, the decrease in IRF3 in the presence of IFITM3 was reversed by treatment of cells with the autophagy inhibitor 3-methyladenine, but not by treatment with the proteasome inhibitor MG132, indicating that IFITM3 promotes IRF3 degradation in autophagosomes. This was further supported by the finding that IFITM3 coimmunoprecipates with the autophagy proteins, Beclin1 and LC3. The authors also observed coimmunoprecipitation of IFITM3 and IRF3, suggesting that these proteins interact, and that IFITM3 may direct IRF3 to autophagosomes. Experiments using cells with genetic deficiencies in autophagosome components in the future will confirm the specific involvement of the autophagy pathway in IFITM3-mediated degradation of IRF3, and will allow the mapping of specific proteins involved in this dampening of the type I IFN response. Overall, this work identifying a feedback inhibition mechanism for the IFN-β induction pathway by IFITM3 highlights the importance and growing appreciation for negative regulators of the type I IFN response during infections.13 This new report has provided insights into a potential biological function for autophagy induction by IFITM3, and also introduces several new research directions that should be exciting areas of investigation in the future. The observed degradation of IRF3 upon expression of IFITM3 did not occur when IFITM1 or IFITM2 was overexpressed, thus identifying a major difference between these highly homologous proteins.12 IFITM2 and IFITM3 share roughly 90% amino-acid identity, and exhibit similar antiviral activities in vitro.6 Thus, it has remained enigmatic that IFITM2 is seemingly unable to compensate for defects in IFITM3 in limiting the severity of influenza virus infections in mice and humans.8,10,11 This new work may suggest that the immunoregulatory role of IFITM3 that distinguishes it from IFITM1 and IFITM2 is critically involved in limiting infection severity in vivo. It will be interesting to map specific amino acids within IFITM3 that mediate interaction with IRF3 and to determine whether these are conserved in IFITM1 and IFITM2. Further, since IFITM3 is regulated by several post-translational modifications, including palmitoylation, ubiquitination and phosphorylation, it will be informative to determine how these IFITM3 modifications impact the degradation of IRF3 and the induction of IFN-β.3,7,14 The discovery of an immunoregulatory role for IFITM3 may also indicate that IFITM3 could have broader effects on immune responses against pathogens beyond the viruses for which it directly blocks membrane fusion. Thus, a wider examination of the role of IFITM3 in immune responses and pathogen susceptibility may be warranted by this exciting new work. Department of Microbial Infection and Immunity, Center for Microbial Interface Biology, The Ohio State University, Columbus, OH 43210, USA Correspondence: Dr JS Yount, PhD, Department of Microbial Infection and Immunity, Center for Microbial Interface Biology, The Ohio State University, 466 W 12th Ave, BRT 790, Columbus, OH 43210, USA. E-mail: yount.37@osu.edu Received: 6 March 2017; Accepted: 6 March 2017 Cellular & Molecular Immunology (2018) 15, 873–874 & 2017 CSI and USTC All rights reserved 2042-0226/17

Opposing roles of endosomal innate immunity proteins IFITM3 and TLR7 in human metapneumovirus infection

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